笔者衷心感谢，并承认医学研究理事会资助的这项研究 - MRC-DPFS金码G1000788-98812。

1.Scaffold制作和消毒<ol><li>溶解的PCL在1,1,1,3,3,3-六氟异丙醇，得到10％w / v的浓度。如所描述的在博斯沃思等人 ，9静电纺丝聚合物溶液（参数:20千伏1毫升/小时，有20厘米）。并收集在旋转的心轴（600转）的边缘排列的纤维。用解剖刀除去收集纤维的色带，然后切成较短的长度 - 3厘米（对于槽和旋转容器）和4厘米（用于细胞培养插入）的长度。 </li><li>用细镊子浸入蒸馏水中个别条和删除。 </li><li>拿着拇指和食指之间的两端;手动拧条，直到它类似于线程。 </li><li>简要地浸没在蒸馏水此线状支架和放置在干净的，非纤维状的卡晾干。 </li><li>一旦干燥，代替单独的干净的微量离心管中，并在蒸馏水中加入1毫升50％体积/体积乙醇。关闭盖子，并留下24小时。 注意:执行下层流下列步骤: </li><li>放置离心管在层流柜和吸出50％的v / v溶液。用1ml 70％体积/体积乙醇蒸馏水，靠近盖更换和离开24小时。 </li><li>重复的90％和在蒸馏水水（1ml体积）100％体积/体积乙醇。 </li><li>洗涤用磷酸盐缓冲盐溶液（PBS）中，每次洗涤24小时，（2×1毫升）脚手架两次。 </li><li>除去PBS中，并添加1 ml的细胞培养介质。注:脚手架已经准备好使用后的24小时。 </li></ol> 2。确定脚手架面积和单元数<ol><li>使用光学显微镜和图像处理软件，测量沿其长度的静电纱线的直径，以确定一个平均值。 </li><li>假设纱是一个圆柱棒和近似使用表面积: </li><li>其中，A =表面积，R =半径和H =长度 注意:表面积计算为18902，800微米2。此外，应该指出的是，实际表面积比该计算更大的纱是由数百个细纤维，这将增加的表面积的。因此细胞的更大数目应能附着到支架上。然而，这并不影响正在测试组之间的直接比较。 </li><li>确定细胞可以通过附着到暴露的支架表面的最大数量: 注意:使用光学显微镜和图像处理软件，人类间质干细胞的直径被确定为20微米（假设细胞是圆的），因此在这种情况下，细胞= 60200的数。 </li></ol> 3.脚手架设置 - 细胞培养插入（图1A） <ol><li>在层流中，打开无菌的6孔细胞培养插入，并从更广泛的环状体中分离的短环带齿。 </li><li>取环带齿朝上。悬垂1所述4厘米支架在环使中心的两侧确保它重叠。取环形体和位置上的带齿环和支架和向下推确保将支架保持在适当的位置，并通过位于细胞培养插管的中心。 </li><li>将细胞培养插管与支架成6孔，低结合板的孔。 </li><li>加入10 mL培养基的脚手架。 </li></ol> 4.脚手架建立 - 槽（图1B） <ol><li>下的层流，代替聚（四氟乙烯）（PTFE）波谷成单独的培养皿和培养介质加入10毫升的低谷。 </li><li>使用镊子披覆3厘米支架进料槽中的一个，并确保其长度位于平行于槽的长边缘。 </li></ol> 5.脚手架建立 - 生物反应器容器（图1C） <ol><li>在层流，将10毫升无菌PBS通过生物反应器容器的主要港口，并留下了10分钟。 </li><li>移除PBS并更换用8ml培养基。 </li><li>使用镊子，经由主端口插入3厘米支架之一到容器。 </li><li>关闭掉的主要港口。 </li></ol> 6.细胞计数<ol><li>从骨髓，根据制造商的协议到通道4之前收获来自培养人间充质干细胞（hMSC的）。 </li><li>从75厘米吸媒体2瓶含有从骨髓（第4代，80％融合）得到的hMSCs。 </li><li>用10ml无菌PBS和抽吸洗细胞。 </li><li>加入3 ml胰蛋白酶孵育烧瓶在37℃，5％的CO 2，直到细胞已经从烧瓶表面脱落。 </li><li>添加7毫升培养基使酶失活，这总体积（10毫升）转移到一个离心管中。 </li><li>通过上下吹打数次重悬该细胞悬浮液，以均匀分散中的细胞媒体和限制细胞团块（10毫升吸管）。 </li><li>取出20微升细胞悬液，转移到一个血球。 </li><li>放置在血细胞计数X10客观光显微镜下的图像。 </li><li>聚焦网格线和计数在网格的每个角落的细胞，在4×4方块数和落入正方形内和那些穿过右手或底部边界线。计数为每组4×4平方（每格4计数）。 </li><li>重复再悬浮和细胞计数三次（步骤6.6-6.9）。 </li><li>计算平均细胞计数，并确定所需的细胞再悬浮（细胞浓度60200在200μl）介质的体积。例如，确定从血球的平均细胞数，然后确定细胞从三个独立计数的总体平均数。由1×10 4乘以此，得到每毫升的细胞数，然后乘以细胞悬浮液的总体积，得到以细胞的数量TAL。使用下面的公式来确定所需的悬浮介质体积: </li><li>离心细胞悬浮液在241×g离心5分钟。 </li></ol> 7.细胞种植<ol><li>从离心管中吸出介质离开细胞沉淀，并替换为所计算出的介质体积。 </li><li>重悬的细胞和培养基对偶数混合。 </li><li>使用P200的吉尔森吸管，缓缓地分配200μl的细胞悬浮液在每个支架通过运行沿该支架的长度和介质液面下的移液管的尖端。离开原状20分钟。 </li><li>对于生物反应器的船只，免除200微升细胞悬液通过主要港口。补足其余2毫升培养经由注射器端口媒体，得到为10ml的总体积。 </li></ol> 8.实验开始<ol><li>转让生物反应器船只到农村信用社，4DQ生物反应器，并设置9的转速旋转。 </li><li>陈德良sfer所述孔板与细胞培养插入和波谷到摇动板，并设置以30rpm旋转。 </li><li>传送孔板与细胞培养插入和波谷至细胞培养箱设定为37℃，5％的CO 2（静态培养）的货架。 </li></ol> 9. DNA检测<ol><li>制备溶液 - 裂解缓冲液，1×TE缓冲液，DNA的标准和细胞DNA工作液 - 按照制造商的说明。 </li><li> 4小时后，从孵化器中取出样品，并在层流放置。 </li><li>所有样品取出介质分数，并放置在单独标示离心管。离心管241 XG。去除上清，加3毫升裂解液。悬浮溶液分手细胞沉淀。 </li><li>使用镊子取下支架并将其放置在装有3毫升裂解液（用于生物反应器内的血管支架离心管，取出支架，用来吸取我之前直径）。用于细胞培养插入物内保持支架，第一释放该支​​架通过切割所述支架附近使用解剖刀刀片的边缘。 </li><li>加入3毫升裂解液到每个支架插座和刮去表面（大力鼓吹的生物反应器的船只）。除去裂解缓冲液，并放置在单独标示离心管中。 </li><li>涡旋每个离心管约1分钟，以确保细胞的足够的搅拌和缓冲，并鼓励细胞膜裂解。 </li><li>在一个黑色的96孔板，加入100微升的裂解液对每个样品部分 - 脚手架，媒体和好（副本）。 </li><li>在黑暗中，加入100微升细胞DNA溶液到含有裂解缓冲液的所有孔中，并轻轻混匀。 </li><li>包括孔用不含DNA和细胞的DNA溶液中以提供一个阴性和阳性对照的孔板裂解缓冲液。 </li><li>使用荧光板阅读器，测量孔的吸光度S使用485nm激发和520 nm发射。 </li><li>与从DNA标准按照制造商的指示生成的标准曲线进行比较的数据。 </li></ol> 10.扫描电子显微镜（SEM）的固定<ol><li>执行以下步骤在层流:4小时后，从他们的插座和地点取出一个新的6孔板（不同孔）内的所有支架。 </li><li>用PBS洗两次支架。 </li><li>执行以下步骤打开板凳上:每孔加入2毫升的PBS 1.5％V / V戊二醛，确保支架完全覆盖。 </li><li>离开板至少30分钟，在4℃，进行细胞固定。 </li><li>除去固定液，用PBS洗两次支架。 </li><li>脱水支架随着乙醇浓度的蒸馏水开始用50％体积/体积，随后70％体积/体积至90％体积/体积。对于每一个浓度，充分浸没在solutio支架N（2ml）中，并离开3分钟。丢弃的解决方案和重复。 </li><li>通过完全浸没在溶液中的支架（2毫升）中，并静置5分钟，脱水在100％乙醇。丢弃的解决方案和重复。 </li><li>化学干燥使用六甲基二（HMDS）在通风橱支架。沉浸在HMDS的支架（2毫升），并留下5分钟。取出HMDS和重复。 </li><li>除去HMDS，并允许支架进行干燥。安装在市售的SEM存根的支架（在此情况下的不锈钢存根与粘合剂碳片）。 </li><li>到缓解的SEM观察中，涂层用金溅射镀膜机进行2分钟的样品，以确保一个薄而均匀的覆盖。 </li><li>内SEM和地方样本使用5千电子伏的电子束可视化细胞接种支架。 </li></ol>

<ol>
	<li>Jauregui, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+to+biomaterials.+The+role+of+several+extracellular+matrix+components+in+the+attachment+of+non-transformed+fibroblasts+and+parenchymal+cells.">Cell adhesion to biomaterials. The role of several extracellular matrix components in the attachment of non-transformed fibroblasts and parenchymal cells.</a> ASAIO transactions/American Society for Artificial Internal Organs. 33 (2), 66-74 (1986).</li><li>Puca, A., Russo, G., Giordano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+Mechano-Transduction+via+Simulated+Microgravity+and+its+Effects+on+Intracellular+Trafficking+of+VEGFR's.">Properties of Mechano-Transduction via Simulated Microgravity and its Effects on Intracellular Trafficking of VEGFR's.</a> Oncotarget. 3 (4), 426 (2012).</li><li>Vincent, L., Avancena, P., Cheng, J., Rafii, S., Rabbany, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulated+microgravity+impairs+leukemic+cell+survival+through+altering+VEGFR-2/VEGF-A+signaling+pathway.">Simulated microgravity impairs leukemic cell survival through altering VEGFR-2/VEGF-A signaling pathway.</a> Annals of biomedical engineering. 33 (10), 1405-1410 (2005).</li><li>Rungarunlert, S., Klincumhom, N., Tharasanit, T., Techakumphu, M., Pirity, M. K., Dinnyes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+Turning+Lateral+Vessel+Bioreactor+Improves+Embryoid+Body+Formation+and+Cardiogenic+Differentiation+of+Mouse+Embryonic+Stem+Cells.">Slow Turning Lateral Vessel Bioreactor Improves Embryoid Body Formation and Cardiogenic Differentiation of Mouse Embryonic Stem Cells.</a> Cellular Reprogramming. 15 (5), 443-458 (2013).</li><li>Wu, X., Li, S. H., Lou, L. M., Chen, Z. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+the+Microgravity+Rotating+Culture+System+on+the+Chondrogenic+Differentiation+of+Bone+Marrow+Mesenchymal+Stem+Cells.">The Effect of the Microgravity Rotating Culture System on the Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.</a> Molecular biotechnology. 54 (2), 331-336 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotating+Microgravity-Bioreactor+Cultivation+Enhances+the+Hepatic+Differentiation+of+Mouse+Embryonic+Stem+Cells+on+Biodegradable+Polymer+Scaffolds.">Rotating Microgravity-Bioreactor Cultivation Enhances the Hepatic Differentiation of Mouse Embryonic Stem Cells on Biodegradable Polymer Scaffolds.</a> Tissue Engineering Part A. 18 (21-22), 2376-2385 (2012).</li><li>Lv, Q., Deng, M., Ulery, B. D., Nair, L. S., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-ceramic+Composite+Scaffolds+for+Bioreactor-based+Bone+Engineering.">Nano-ceramic Composite Scaffolds for Bioreactor-based Bone Engineering.</a> Clinical Orthopaedics and Related Research. 471 (8), 2422-2433 (2013).</li><li>Hammond, T. G., Hammond, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+suspension+culture:+the+rotating-wall+vessel.">Optimized suspension culture: the rotating-wall vessel.</a> American Journal of Physiology-Renal Physiology. 281 (1), F12-F25 (2001).</li><li>Bosworth, L. A., Alam, N., Wong, J. K., Downes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+2D+and+3D+electrospun+scaffolds+intended+for+tendon+repair.">Investigation of 2D and 3D electrospun scaffolds intended for tendon repair.</a> Journal of Materials Science: Materials in Medicine. 24 (6), 1605-1614 (2011).</li><li>Dormer, N. H., Qiu, Y., Lydick, A. M., Allen, N. D., Mohan, N., Berkland, C. J., Detamore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+human+bone+marrow+stromal+cells+in+hydroxyapatite-loaded+microsphere-based+scaffolds..">Osteogenic differentiation of human bone marrow stromal cells in hydroxyapatite-loaded microsphere-based scaffolds..</a> Tissue Engineering Part A. 18 (7-8), 757-767 (2011).</li><li>Rayatpisheh, S., Heath, D. E., Shakouri, A., Rujitanaroj, P. O., Chew, S. Y., Chan-Park, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+cell+sheet+technology+and+electrospun+scaffolding+for+engineered+tubular,+aligned,+and+contractile+blood+vessels.">Combining cell sheet technology and electrospun scaffolding for engineered tubular, aligned, and contractile blood vessels.</a> Biomaterials. 35 (9), 2713-2719 (2014).</li><li>Wismer, N., Grad, S., Fortunato, G., Ferguson, S. J., Alini, M., Eglin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+electrospun+scaffolds+for+annulus+fibrosus+tissue+engineering:+effect+of+scaffold+structure+and+composition+on+annulus+fibrosus+cells+in+vitro.">Biodegradable electrospun scaffolds for annulus fibrosus tissue engineering: effect of scaffold structure and composition on annulus fibrosus cells in vitro.</a> Tissue Engineering Part A. (3-4), 672-682 (2014).</li><li>Yavin, E., Yavin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+and+culture+of+dissociated+cells+from+rat+embryo+cerebral+hemispheres+on+polylysine-coated+surface.">Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface.</a> The Journal of cell biology. 62 (2), 540-546 (1974).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guan,+of+focal+adhesion+kinase+with+its+potential+substrate+phosphatidylinositol+3-kinase.">Guan, of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase.</a> Proceedings of the National Academy of Sciences. 91 (21), 10148-10152 (1994).</li><li>Grant, D. S., Tashiro, K. -. I., Segui-Real, B., Yamada, Y., Martin, G. R., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+different+laminin+domains+mediate+the+differentiation+of+human+endothelial+cells+into+capillary-like+structures+in+vitro.">Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro.</a> Cell. 58 (5), 933-943 (1989).</li><li>Carrier, R. L., Papadaki, M., Rupnick, M., Schoen, F. J., Bursac, N., Langer, R., Freed, L. E., Vunkaj-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+engineering:+cell+seeding,+cultivation+parameters,+and+tissue+construct+characterization.">Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.</a> Biotechnology and bioengineering. 64 (5), 580-589 (1999).</li></ol>

The authors declare that they have no competing financial interests.

从生物聚合物制成电纺纤维基质经常用于支持细胞附着和增殖为生物材料和/或组织工程应用11,12。在这些情况下，基质常常薄板纤维容易覆盖细胞培养井板的整个基础，从而在与接种的细胞可提高细胞附着完全接触的。然而，如果生物材料支架不完全覆盖所述孔板的底部，有一个高的机会，该接种的细胞的相当大的比例不会停留在与支架接触，并最终将不能附着。该研究调查了几种不同的方法对种子细胞到支架不覆盖孔板的底部，以确定可能被推荐用于未来的基于细胞的实验的优化技术。 五个不同的调校进行了研究（ 图1）:scaffo使用低绑定孔板内的细胞培养插入LDS（静电纱）举行，要么保持静态条件下或摇动30转;支架置于狭窄的PTFE槽内，并保持静态或动摇在30转;和安置生物反应器内旋转的船只9转支架。确定细胞，通过DNA分析附着在静电纱线的数量显示出附件的所有接种设置窗口（ 图2）低的百分比;这进一步从扫描电子显微照片（SEM）（ 图3）确认。最大的细胞附着 - 30％〜18060细胞-was观察举行了细胞培养插入内，进行连续运动的纱线。有趣的是，最低细胞附着（15％），用于通过细胞培养插入持有，但在静态条件下保持的纱线，这将表明，包含径向运动对保持细胞与支架接触的积极效果达到了。然而，但是应当注意的是，媒体的流量连续旋绕可能负责从SEM图像中观察到的细胞团块。该摇动板被设置在它的最低设置 - 30rpm下 - 这可能是一个限制到这种设置。使用较慢的径向运动可有助于预防或减少细胞凝集，也可以提高细胞附着作为细胞将经历较小的力。未来的实验应该着力优化的理想摇床速度提高细胞附着。掺入运动对于保持槽内没有导致了类似的趋势纱线，与这两种情况下，得到18％的附着性（〜10836个细胞）;虽然这可能是由于在槽内的支架的部分浮起（观察放置在摇动器板上的槽），因为它们并没有固定在基座上。支架的局部浮将防止已经从进入与材料和粘附接触下沉到槽的底部的任何细胞。在t他的特殊设置中，该槽被装在一个培养皿中，并共10毫升体积的介质中加入。槽的尺寸小意味着大多数媒体内的培养皿本，如果有任何的运动，细胞可能漂离槽到培养皿并保持完全脱离支架接触。为了克服这些限制，进一步的实验应该包括在该协议中，从而使支架的端部被固定到用无菌细针槽的底部的额外步骤，因为这应当防止其浮起和运动（特别是对于暴露于支架径向运动），这最终应导致附接至所述支架单元的数量增加。细胞的16％的已附连在旋转容器内存在的纱线。尽管是一个行之有效的技术为3D的文化，并出现从船只的主要港口拆除支架，这可能导致松散ATTAC问题丢失HED细胞。容器可以完全打开将消除这个问题;这些是可以购买的，但都显着高于在该研究中使用的一次性容器更昂贵。 这项研究表明，目前的问题播种支架不包括标准的细胞培养孔板的整个基。播种一个已知的细胞数目导致小于三分之一附着到所述支架，尽管支架的表面积，允许所有的细胞附着。这可能在其他基于细胞的试验，可能评估生物相容性和细胞材料/细胞 - 细胞的行为和相互作用与该支架作为一个潜在的未来医疗装置的有害后果。该研究的进一步限制可能包括4小时的时间点-尽管是足够长的时间，以确保初始细胞接种（细胞已被证明牢固附着到基板30分钟13,14,15内），它可能是合理的，在vestigate稍后的时间点，提供细胞较长的时间帧期间不增殖，因为这本来歪斜的起始细胞数。还原的介质体积，在此情况下10毫升，还可以改善细胞和支架之间的接触，并最终增加细胞附着。未来的研究也应考虑细胞活力作为细胞接种的过程中可能会导致细胞损伤和/或细胞死亡16。细胞的DNA分析不可行的和非存活细胞区分，因为这样的活/死测定法，例如，将突出生存能力的水平。 本次调查提高认识到细胞附着在支架的实际数量，尽管播种一个已知量。对于依赖于细胞的起始数的研究，这是非常重要的是，研究人员知道究竟有多少该数字做实际上附着于感兴趣的底物。

支架例行正在开发和研究的生物材料和组织工程应用。因此，它们通常被接种细胞及其在体外行为通过确定细胞增殖和细胞数，例如测定法表征。进行实验，如这些，当务之急是初始小区数量是已知的，并研究经常述在每毫升或厘米2细胞的数目而言播种浓度。虽然这是很好的做法，特别是对于按比例放大的目的，不考虑细胞附着在支架的表面（其也依赖于生物材料表面1的粘结性）的实际数量。这对于支架不覆盖细胞培养井板的整个基更是如此作为细胞可以从构建体都消失，而且，由于实验的通常的静态性质，可能永远不会回到与英特的材料接触休息。电纺纤维纱是一个支架未覆盖的井（ 图1A）的基部的一个很好的例子。在这种情况下，低结合孔板，但没有经表面处理的应用，以防止细胞附着在板的表面，因此扭曲的任何公基测定法的结果。 孔板容易地用于细胞接种到支架的，但它们不是唯一可用的方法。旋转细胞培养系统，一种生物反应器由生命科学部于上世纪80年代开发的NASA，同样可以用于在与模拟微重力三维（3D）环境种子支架。这种类型的生物反应器仍然是一个流行的选择与世界各地的研究人员和研究中已经纳入了细胞信号2,3，干细胞4,5和组织工程6,7。是什么让旋转生物反应器最好孔板是维修的3D环境中，这有助于防止分化的细胞，从去分化，这是常有的情况下，当常规的2D条件8内进行培养。 本文研究不同的技术来作为制作博斯沃思等人 ，9，以便最大限度地附连到这些支架一个4小时期间内的细胞的初始数量上静电聚（ε -己内酯）纤维丝接种人类间质干细胞。对于2D培养，纱线牢固孔板或定做的聚（四氟乙烯）（PTFE）波谷内保持与静态条件下保持，或摇动以30rpm。对于3D的文化，纱线和细胞生物反应器旋转的船只在9转速范围内举行。

<table><tbody><tr><td>Distilled water</td><td>in-house supply</td><td>n/a</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1117271000</td><td></td></tr><tr><td>Phosphate Buffered Saline solution</td><td>Life Technologies</td><td>70013016</td><td></td></tr><tr><td>Human mesenchymal stem cells</td><td>PromoCell GmbH</td><td>C-12974</td><td></td></tr><tr><td>MSC culture media</td><td>PromoCell GmbH</td><td>C-28010B</td><td>Warmed to 37 oC before use</td></tr><tr><td>Supplement mix</td><td>PromoCell GmbH</td><td>C-39810</td><td>Add to culture media</td></tr><tr><td>Antibiotic/antimyotic mix</td><td>Sigma-Aldrich</td><td>A5955</td><td>Add to culture media</td></tr><tr><td>Trypsin (0.05%) EDTA (0.02%)</td><td>Sigma-Aldrich</td><td>59417C</td><td>Warmed to 37 &amp;#176;C before use</td></tr><tr><td>Cell culture flasks (T75)</td><td>Becton Dickinson Ltd</td><td>353110</td><td></td></tr><tr><td>Low binding 6-well plates</td><td>Costar Corning</td><td>3471</td><td></td></tr><tr><td>6-well CellCrowns</td><td>Scaffdex</td><td>C00003S</td><td></td></tr><tr><td>Petri-dish 50 ml deep</td><td>Sterilin</td><td>124</td><td></td></tr><tr><td>PTFE troughs</td><td>in-house production</td><td>n/a</td><td></td></tr><tr><td>Disposable RCCS vessels 10 ml</td><td>Synthecon</td><td>D-410</td><td></td></tr><tr><td>4 Vessel Rotary Cell Culture System bioreactor</td><td>Synthecon</td><td>RCCS-4DQ</td><td></td></tr><tr><td>Shaker plate</td><td>Stuart</td><td>SSM1</td><td>Mini Orbital Shaker</td></tr><tr><td>Haemocytometer</td><td>Digital Bio</td><td>DHC-F01</td><td>Disposable C-Chip</td></tr><tr><td>Centrifuge tube</td><td>Deltalab</td><td>352096, 429901</td><td>15 ml and 50 ml</td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Rotafix 32 A</td><td></td></tr><tr><td>Syringe 3 ml</td><td>Shield Medicare Ltd</td><td>3039820</td><td></td></tr><tr><td>Pipettes</td><td>Sterilin</td><td>40305, 47310, 40125</td><td>5, 10 and 25 ml</td></tr><tr><td>Gilson pipettes</td><td>SLS</td><td>F144801, F144802, F123600, F123601, F123602</td><td>P2 - P1000</td></tr><tr><td>Pipette tips</td><td>SLS</td><td>PIP7852, PIP7834, PIP7840</td><td></td></tr><tr><td>Micro test tube 1.5 ml</td><td>Eppendorf</td><td>30125.15</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>9002-93-1</td><td></td></tr><tr><td>PicoGreen Assay</td><td>Invitrogen</td><td>P7589</td><td>Assay set-up in the dark</td></tr><tr><td>Black 96-well plate</td><td>Greiner Bio One</td><td>655086</td><td></td></tr><tr><td>Fluorescent plate-reader</td><td>BGM Labtech</td><td>FLUOstar Optima</td><td></td></tr><tr><td>Glutaraldehyde 25%</td><td>TAAB Laboratories</td><td>G002</td><td>Made to a concentration of 1.5% v/v in PBS</td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Sigma</td><td>999-97-3</td><td></td></tr><tr><td>Aluminium stubs (SEM)</td><td>Agar Scientific</td><td>G301</td><td></td></tr><tr><td>Carbon tabs (SEM)</td><td>Agar Scientific</td><td>G3347N</td><td></td></tr><tr><td>Gold sputter coater</td><td>Edwards</td><td>S150B</td><td></td></tr><tr><td>Scanning Electron Microscope (SEM)</td><td>Phenom World</td><td>Phenom Pro</td><td></td></tr></tbody></table>

optimizing attachment of human mesenchymal stem cells on poly(&#949;-caprolactone) electrospun yarns

研究生物材料和组织工程通常包括基于细胞的体外研究，这需要的起始细胞数初始知识。尽管研究人员通常引用其接种密度，这并不一定表示已附着于有问题的材料的细胞的实际数量。这是特别为材料或支架，其不包括标准的细胞培养孔板的底部的情况。本研究探讨接种在一个已知的数上静电纺丝的聚（ε - 己内酯）纱线在培养4小时后，自人骨髓间充质干细胞的初始附着。静电纱内几个不同的调校举行，包括生物反应器容器旋转9的转速，位于低结合孔板和聚四氟乙烯（PTFE）的槽放置在培养皿中的细胞培养插入物。后两者经受静态条件或定位在振荡器上板（30 <spañ风格="字体大小:14px的;线高度:28px;"&gt; RPM）。后4小时在37℃，5％的CO 2中 ，接种细胞的位置是由细胞的DNA分析测定。从容器中取出，放入裂解液支架。媒体馏分类似地除去并离心 - 上清液丢弃，沉淀捣碎用裂解缓冲液。裂解缓冲液加入每个容器，或良好，并且刮下以释放任何细胞可以存在。细胞的DNA分析测定细胞在支架内时，媒体和井馏分的百分比。细胞附着率较低为所有的实验室设备，具有最大的附着（30％）的细胞培养插入物内保持并进行摇动运动的纱线。这项研究提出认识到附加到脚手架不管通过上述细胞接种密度的细胞的实际数量。

该结果强调以下4小时后播种每种实验设置的细胞的位置的影响。 图2A表明细胞在此期间已附着在支架的表面的百分比。 8.5皮克/细胞A转换因子是用来测量的DNA含量为细胞数转换，从而确定电池10的比例。对于所有的播种调校研究，细胞附着的百分比相对较低，具有最大的细胞粘附（30％）的细胞培养插入物内保持并摇动以30rpm（插入振荡器）的支架。坚持最低（15％）是在细胞培养插入内举行脚手架，并保持静态条件（将静态）下。 大量细胞是媒体级分（ 图2B）内时，最明显的是对于低结合板（插入静态）和旋转式容器内保持细胞培养插入物为50％和51％的分别。槽内保持支架证明细胞保持器本身内本的凸起数 - 48％的细胞为槽振荡器和用于槽的静态的50％。 扫描电子显微镜所允许的细胞接种支架的视觉评估（ 图3）。代表图像强调不论接种建立细胞的有限的存在在纤维表面上，。然而，更大数目的细胞和细胞聚集体是存在于细胞培养插入物内保持并摇动以30rpm（插入振荡器）的支架。 <img alt="图1" src="/files/ftp_upload/52135/52135fig1.jpg" /> 图1.实验调校，其中静电纺丝纱内举行; （A）的细胞培养物的插入和低结合孔板，（B）聚（四氟乙烯）（PTFE）和波谷培养皿;和（C）生物反应器容器。 <a href="https://www.jove.com/files/ftp_upload/52135/52135fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52135/52135fig2.jpg" /> 图2.位置细胞4小时后播种到PCL电纺的纱线采用不同播种调校的。 （A）的演示已经附连到的PCL支架的细胞的百分比（平均值±标准偏差）;（B）的突出位置细胞的百分比传播三个馏分内-媒体，以及与支架（n = 4时，数据表示为平均值）。 <a href="https://www.jove.com/files/ftp_upload/52135/52135fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" sRC ="/文件/ ftp_upload / 52135 / 52135fig3.jpg"/&gt; 图3.代表性的扫描电子显微镜对PCL电纺的纱线与人骨髓间充质干细胞，采用不同的实验调校初始播种后4小时 （以1,000倍的放大倍率的所有图像，比例尺= 130微米。） <a href="https://www.jove.com/files/ftp_upload/52135/52135fig3highres.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a>

Watch this Scientific Journal Video about Optimizing Attachment of Human Mesenchymal Stem Cells on PolyÃŽÂµ-caprolactone Electrospun Yarns at JoVE.com

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

optimizing-attachment-human-mesenchymal-stem-cells-on-poly

Research

JoVE Journal

Bioengineering

9.7K 次观看.  The University of Manchester. This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

视频: Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε-caprolactone) Electrospun Yarns

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

三层次的静电，以模仿母语动脉结构采用聚己内酯，弹性和胶原的初步研究

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

科研

生物工程

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

静电基础：优化解决方案和设备参数

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

生物学

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.